Lactoferrin (Lf), an 80 kD glycoprotein that can bind two atoms of Fe(lll), is a major protein in human milk, and also present in exocrine secretions, e.g. pancreatic fluid and bile, and in neutrophils. Lf has been suggested to have several functions, including a role in intestinal Fe absorption, reproductive function, antimicrobial action, cellular proliferation and immune competence. However, although some support for such activities have been obtained, little is known about the mechanisms by which Lf exerts these functions. We previously showed the existence of Lf receptors (LfR) in human small intestine by kinetic binding studies. We have recently cloned and expressed human and mouse LfR, which are expressed in several tissues. Transfection of human intestinal cells showed increased uptake of iron and of Lf. In the proposed project we intend to determine the tertiary structure of LfR and the Lf-LfR complex by X-ray diffraction to better understand the organization of the LfR molecule and its interaction with Lf. We will also determine the specific site of the Lf molecule that binds to the LfR by using truncated versions (N-lobe, C-lobe) expressed as chimeras with transferrin in baculovirus. Peptides binding to the LfR will be generated by combinatorial chemistry. Oligonucleotide probes will be used for in situ hybridization and an LfR antibody will be used for immunostaining of human and mouse tissues, with particular emphasis on the small intestine and mouse embryonic development. Pathways of Lf internalization will be studied by inhibitors specifically inhibiting coated pits and caveolae-mediated pathways, respectively. Cellular responses to Lf mediated by the LfR will be studied by microarrays for signal transduction pathways. The effect of Fe, lipopolysaccharide (LPS) and nitric oxide on LfR expression will be studied by using several cell lines. We will also explore the biological significance of the LfR by using a conditional knockout mouse. We will clone the mouse LfR gene and construct a targeting vector in which the first exon is flanked by two Iox P sites. We will then use inducible cre transgenic mice to produce LfR knockouts in various tissues, such as small intestine and mammary gland, as well as at various stages of development. Basic properties of the knockouts will be compared with those of the wild type. Possible observations in the knockouts will be impaired reproduction and immune competence, decreased levels of Lf in milk, developmental abnormalities, lower Fe status of pups and diminished protective effect of Lf against LPS-caused mortality. Overall, our understanding of the physiological significance of Lf and its receptor will be increased.